Treatment of type 1 immune response-mediated inflammatory lung disease by modulation of ifn-gamma activity

ABSTRACT

The present invention relates to preventing, or treating and/or reducing the severity or progression of Type 1 immune response-mediated inflammatory lung disease. More particularly, the present invention provides a method for preventing or treating chronic obstructive pulmonary disease (COPD), severe asthma, sarcoidosis, berylliosis or cystic fibrosis by neutralizing or reducing IFNγ bioactivity which can be achieved either by in vivo administration of IFNγ neutralizing molecules or by in vivo immunization with pharmaceutical compositions comprising immunogenic IFNγ proteins or IFNγ-derived (poly)peptides or their corresponding nucleic acid sequences.

FIELD OF THE INVENTION

The present invention relates to preventing, or treating and/or reducingthe severity or progression of Type 1 immune response-mediatedinflammatory lung disease. More particularly, the present inventionprovides a method for preventing or treating chronic obstructivepulmonary disease (COPD), severe asthma, sarcoidosis, berylliosis orcystic fibrosis by neutralizing or reducing IFNγ bioactivity which canbe achieved either by in vivo administration of IFNγ neutralizingmolecules or by in vivo immunization with pharmaceutical compositionscomprising immunogenic IFNγ proteins or IFNγ-derived (poly)peptides ortheir corresponding nucleic acid sequences.

BACKGROUND ART

The present invention relates to preventing the onset of symptoms,treating and/or reducing the severity or progression of COPD and otherType 1 immune response-mediated (T1) inflammatory lung diseases such as,but not limited to, severe asthma, sarcoidosis, berylliosis, and cysticfibrosis. T1 inflammatory lung diseases are characterized by a Type 1immune response mediated by T helper-1 cells (CD4+) and T cytotoxic-1cells (CD8+) and by increased production of interferon gamma (IFNγ),tumor necrosis factor (TNF), and interleukin-2 (IL-2). T1 cytokinesevoke cell-mediated immunity characterized by prominent lung tissueinfiltration of macrophages, neutrophils, and T-cells.

Severe Asthma

Asthma is a disease of the respiratory system characterized byhyper-responsiveness to bronchoconstricting stimuli, inflammation, andchanges in respiratory epithelium Asthmatic patients in whom the diseaseprocess is either refractory to therapy or requires persistent use ofhigh dose systemic anti-inflammatory corticosteroids in order tomaintain reasonable control of symptoms are referred to in literature aspatients suffering from severe or irreversible or refractory asthma(Kaplan N. M. et al., 2000). Patients with severe persistent asthma havecontinual symptoms, frequent exacerbations, frequent nighttime symptomsand evidence of severe obstructive lung disease on pulmonary functiontesting (Forced Expiratory Volume in one second: FEV1<60%). Thenarrowing of airways causes ventilation perfusion imbalance, lunghyperventilation, and increased work of breathing that may lead toventilatory muscle fatigue and life-threatening respiratory failure(Papiris S. et al., 2002). WO 01/34180 by Block Lutz-Henning describesthe use of IFNγ for the treatment of severe asthma broncliale. Neweffective treatments are needed for this subpopulation of asthmaticpatients (Stirling R. G. and Chung K. F., 2001).

Increased numbers of neutrophils are observed in the lung mucosa and thebronchoalveolar fluid of severe asthmatics (Wenzel et al., 1997).Eicosanoid mediators such as thromboxane and leukotriene B4 are alsohigh in the lung tissue of these patients. No difference in eosinophilconcentration is observed in BAL fluid derived from healthy controls orfrom severe asthmatics.

Severe asthma is differentiated from mild/moderate asthma by distinctinflammatory processes involving varied cytokine expression profilesand/or effector cells. In contrast to severe asthmatics, mild ormoderate asthmatics show enhanced concentrations of eosinophils in theirlungs and a Type 2 inflammation characterized by predominantinfiltration of T helper-2 T lymphocytes that produce IL-4, IL-5, IL-9and IL-13. Neutrophils are absent Symptoms in mild to moderateasthmatics are well controlled by treatment with β2-agonists andcorticosteroids.

Sarcoidosis

Sarcoidosis and berylliosis are interstitial lung diseases. Theinterstitium (the space between the tissues) of the lungs includesportions of the connective tissue of the blood vessels and air sacs.Interstitial lung diseases begin with inflammation of the lung cells.The lungs stiffen as a result of inflammation of the air sacs(alveolitis) and scarring (fibrosis) (he Lungs in Health and Disease,National Heart, Lung and Blood Institute; NIH Publication No. 97-3279,August 1997). Sarcoidosis is a systemic granulomatous disease of unknownaetiology and world-wide distribution. It most commonly affects youngadults and presents as with bilateral hilar lymphadenopathy, pulmonaryinfiltration, reticuloendothelial involvement, eye and skin lesions.Shortness of breath (dyspnea) and a cough that will not go away can beamong the first symptoms of sarcoidosis (Sarcoidosis, National HeartLung, and Blood Institute; NIH publication No. 95-3093, reprinted July1995). The hallmark of pulmonary sarcoidosis is a mononuclear alveolitiswhich is characterized by activated CD4+lymphocytes,monocytes/macrophages, and non-caseating granulomas. An imbalance in theexpression of T1 and T2 cytokines by alveolar cells is thought to playan important role in the immunopathogenesis of sarcoidosis. It is wellestablished that T1 cytokines are important mediators in pulmonarysarcoidosis and that there is a dependence of granulomatous inflammationon T1 cytokines. Alveolar cells spontaneously release the T1 cytokinesIFNγ and IL-2 but not T2 cytokines. However, it is suggested that thecytokine patterns change during the course of the disease (Möllers M.,et al., 2001). Shigehara K. et al., 2001, demonstrated that IL-12 andIL-18 were increased in BAL fluids of patients with sarcoidosis. IL-12and IL-18 drive the immune response in to Type 1 direction.

In recent years, new therapies have been studied for sarcoidosis. Drugsused to treat patients with sarcoidosis are corticosteroids, cytotoxicagents, immunomodulators (chloroquine and hydroxychloroquine) and theantileprosy drugs clofazimine and minocycline. A key cytokine in chronicsarcoidosis appears to be TNF. Drugs that inhibit its release or blockits bioactivity such as pentoxifylline and thalidomide have beenreported to be effective for treatment of sarcoidosis (Baughman R. P.,2002).

Berylliosis

Berylliosis or Chronic Beryllium Disease (CBD) is an environmentalchronic inflammatory disorder of the lungs caused by inhalation ofinsoluble beryllium (Be) dust and characterized by the accumulation ofCD4+T cells and macrophages in the lower respiratory tract According tothe type and level of Be exposure, the reactions vary from acutetracheobronchitis, chemical pneumonitis, and metal fume fever to achronic granulomatous lung disorder. Lung T cells respond to berylliumwith the release of T1 cytokines such as IL-2, the migration inhibitorfactor (MIF, IFNγ, and TNF-α. Furthermore, a positive association with aHLA class II allele, HLA-DP, has been described (Saltini C. et al.,2001). Most patients treated with corticosteroids need to remain ontherapy for life (Rossman M. D., 2001).

Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive disorder caused by nearly1000 different mutations of the cystic fibrosis transmembraneconductance regulator (CFMI) gene. It is a multisystem disordercharacterized by defective electrolyte transport in epithelial cells andabnormally viscous mucus secretions from glands and mucus epithelia.Symptoms are pancreatic insufficiency (PI) associated with neonatalmeconium ileus and chronic obstructive lung disease superimposed withrecurrent opportunistic infections that progressively destroy lungtissue. The inflammatory response is a primary cause of irreversiblelung damage. Inflammation is present in CF patients and precedes thebacteria infections, as demonstrated by increased levels ofneutrophils,.T and IL-8. Other complications,include liver disease,chronic sinusitis, infertility in male patients and elevated sweatchloride concentrations. Despite advances in genomic technologies anddrug discovery, drug therapy often improves disease symptoms but doesnot cure the disease. One of the main causes of this failure to cure CFmay be attributable to genetic variability and to the scarce knowledgeof CF biochemistry. The development of new treatments may be importantfor the life expectancy of patients. Current CF therapeutic strategiesinclude lung transplantation, antimicrobial treatment, corticosteroids,non-steroidal anti-inflammatory drugs like ibuprofen, ion channeltherapy, protein-assist therapy, and gene therapy (Sangiuolo F. et al.,2002).

COPD

Chronic obstructive pulmonary disease (COPD) is currently the sixthleading cause of death and the 12th leading cause of morbidityworld-wide. By the year 2020, COPD is expected to be the third leadingcause of death and the fifth leading cause of disability.

COPD is a disease state characterized by chronic and slow progressivedevelopment of airflow limitation that is not fully reversible sad ispunctuated by episodic exacerbations due to viral or bacterialinfections. The airflow limitation is associated with an abnormalinflammatory response of the lungs to noxious particles or gases(Executive Summary, Global Strategy for the diagnosis, management andprevention of chronic obstructive pulmonary disease; NHLBI/WHO WorkshopReport). COPD comprises chronic bronchitis, chronic obstructivebronchiolitis and emphysema (Ockie M. J. et al., 2000). Conic bronchitisis defined as the occurrence of coughing and by production of sputum onmost days for at least 3 months over 2 consecutive years. Emphysema ischaracterized by destructive enlargement of airspaces with loss ofnormal architecture and lung elasticity.

Cigarette smoking is the dominant factor for the development andprogression of COPD. However, only 15% of smokers develop COPD and >15%of COPD-related mortality occurs in people who have never smoked,suggesting that other factors are important Genetic factors such asα₁-antitrypsin deficiency, resulting in enhanced neutrophil elastaseactivity, account for 2% of emphysema patients and polymorphism of theTNF-αgene, leading to enhanced TNF production, may also play animportant role. The role of infections in both the development andprogression of COPD is getting increased attention, including adenoviraland rhinoviral infections in patients with emphysema. Occupational andenvironmental exposures to various pollutants are also considered to beimportant factors in the development of COPD. (Mannino D. M. et al.,2002; Barnes P. J., 2000).

A diagnosis of COPD should be considered in any patient who has symptomsof cough, sputum production, or dyspnea, and/or a history of exposure torisk factors for the disease inhaled gases (nitric oxide and carbonmonoxide) and inflammatory markers in exhaled breath condensate can beused as non-invasive markers of COPD (Leckie M. J. et al., 2000). Thediagnosis is confirmed by spirometry. The presence of apost-bronchodilator FEV₁<80% of the predicted value in combination withan FEV₁/FVC <70% confirms the presence of airflow limitation that is notfully reversible (FEV1=forced expiratory volume in one second; FVC=forced vital capacity).

A simple classification of disease severity into four stages isrecommended. All FEV₁values refer to post-bronchodilator FEV₁.

-   -   Stage 0: At Risk: characterized by chronic cough and sputum        production. Lung function, as measured by spirometry, is still        normal.    -   Stage I: Mild COPD: characterized by mild airflow limitation        (FBV₁/FVC<70% but FEV₁80% predicted) and usually, but not        always, by chronic cough and sputum production.    -   Stage II: Moderate COPD: characterized by worsening airflow        limitation (30% ≦FEV₁<80% predicted) and usually the progression        of symptoms, with shortness of breath typically developing on        exertion This is the stage at which patients typically seek        medical attention because of dyspnea or an exacerbation of their        disease. The division into stages IIA and IIB is based on the        fact that exacerbations are eely seen in patients with an FEV₁        below 50% predicted The presence of repeated exacerbations has        an impact on the quality of life of patients and requires        appropriate management    -   Stage III: Severe COPD: characterized by severe airflow        limitation (FEV₁<30% predicted) or the presence of respiratory        failure or clinical signs of right heart failure. Patients may        have severe (Stage III) COPD even if the FEV₁ is >30% predicted,        whenever these complications are present. At this stage, quality        of life is appreciably impaired and exacerbations may be        life-threatening.

COPD is generally regarded as a separate condition from asthma(reversible airflow limitation) in terms of inflammatory processes,underlying pathology, and responses to treatment Airway inflammation inasthma is characterized by infiltration of eosinophils and T helper-2lymphocytes. Macrophages are less frequent and CD8 T cells are usuallyabsent The T2 cytokines predominate: IL-4 and IL-13 play an importantrole in IgE production, whereas IL-5 is critical for eosinophil growthand differentiation (Barnes P. J., 2000).

Airway inflammation in COPD is characterized by the presence ofneutrophils, macrophages and CD8 T cells. Histopathological studies showthat most inflammation in COPD occurs in the peripheral airways(bronchioles) and lung parenchym. The bronchioles are obstructed byfibrosis and there is destruction of lung parenchym. Bronchial biopsyresults show similar find There is also a marked increase in macrophagesand neutrophils observable in bronchoalveolar lavage fluid and inducedsputum.

Emphysema is caused by an imbalance of proteases and proteaseinhibitors. The concentration of inflammatory mediators such asleukotriene B₄, TNF-α and IL-8 are increased in sputum of patients withCOPD (Barnes P. J., 2000). Other inflammatory cytokines associated withCOPD are TGF-β, IL-1, IL-6, IL-11 and IL-18. Also a role for IFNγ inCOPD has been mentioned in the literature, however with some differingdata Wang Z. et al. (2000) describe a transgenic mouse model, with IFNγinducibly targeted to the adult murine lung, showing emphysema,macrophage and neutrophil infiltration and inversedproteasetanti-protease ratio. Majori M. et al.(1999) showed an increasein the percentage of IFNγ-producing cells among peripheral blood Thelper cells from patients with COPD. Analysis of cytokine levels in BAL(bronchoalveolar lavage) discloses a prevalent T1 cytokine pattern inCOPD, however this difference was not significant when COPD patientswere compared with the other two groups (asthmatics and non-smoker)(Balbi B. et al., ATS 2002). Lethbridge M. W. G. et al. (2002) evenshowed a disproportionately low number of IFNγ-expressing airwaylymphocytes in COPD smokers as compared to healthy smokers and healthyex-smokers. The association of chronic inflammation with thepathophysiology of COPD makes IL-1, IL-18, and TNF-αtargets fortherapeutic intervention (de Boer W. L., 2002). Macrophages predominateand appear to play a central role as they have the capacity to produceall the pathologic changes of COPD (Hautamaki, 1997). Macrophagesrelease LTB4 and 1L-8, which are potent neutrophil chemo-attractants,and multiple proteases responsible for the continued proteolyticactivity in the lungs of patients with emphysema.

At the present time, there are no known drugs that slow the relentlessprogression of COPD and there is a pressing need to develop new drugs tocontrol the inflammatory and destructive processes that underlie thedisease. Smoking cessation is the only measure that will slow theprogression of COPD. However, even if the patient stops smoking, thedamage already caused will continue to cause symptoms (Banes P. J.,2001). Currently available drugs that provide symptomatic relief in COPDare:

-   -   Bronchodilators, which are the mainstay of current drug therapy        for COPD (Leckie M. J. et al., 2000).    -   Inhaled corticosteroids are also widely prescribed for COPD but        have a risk of systemic side effects (Barnes P. J., 2000). The        usefulness of inhaled corticosteroids in COPD remains        controversial (Leckie M. J. et al., 2000).    -   Theophylline    -   Although antibiotics are still widely used for exacerbations of        COPD, it is increasingly recognized that exacerbations may be        due to vial infections of the upper respiratory tract or may be        noninfective, so that antibiotic treatment is not always        warranted (Barnes P. J., 2002).

Nonpharmarcologic treatments include oxygen therapy, non-invasiveventilation, exercise training, pulmonary rehabilitation and lung volumereduction surgery (Barnes P. J., 2000).

A better understanding of the cellular and molecular mechanisms involvedin COPD provides new molecular targets for the development of drugs andseveral classes of new drugs are now under development Leukotriene B₄(LT B₄) inhibitors, chemokine inhibitors, TNF-α inhibitors,antioxidants, iNOS inhibitors, and corticosteroids are examples ofinflammatory mediator antagonists. Examples of protease inhibitors areneutrophil elastase inhibitors, cathepsin, inhibitors, α₁-antitrypsin,secretory leukoprotease inhibitor and elafin New anti-inflammatory drugsfor COPD are PDE type IV inhibitors, NF-κB inhibitors, adhesion moleculeblockers, IL-10, p38 MAP kinase inhibitors, and P13-kinase inhibitors(Banes P. J., 2001).

As described earlier, currently available therapies for T1 inflammatorylung disease are broad acting and directed to the improvement ofclinical symptoms and/or to a general reduction of inflammation. Thereis thus a need for new selective therapeutic strategies which end therelentless progression of said diseases by targeting a key mediator ofthe underlying mechanism.

Notwithstanding the fact that several potential therapies for T1mediated inflammatory lung disease have been proposed, no prior artexists revealing that neutralizing IFNγ bioactivity is effective in thetreatment of T1 mediated inflammatory lung diseases such as COPD, severeasthma, sarcoidosis, berylliosis, and cystic fibrosis.

The present invention demonstrates that the inflammatory and destructiveprocesses that underlie T1 inflammatory lung diseases can be treated byneutralizing IFNγ.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods andcompositions for preventing or treating T1 inflammatory lung disease,particularly COPD, emphysema, chronic bronchitis, bronchiolitis, severeasthma, sarcoidosis, berylliosis, and cystic fibrosis.

The present invention provides a method for preventing or treating T1inflammatory lung disease, said method comprising reducing orneutralizing the bioactivity of IFNγ. Several methods and compositionscan be applied for this and are thus part of the current invention.

An aspect of the present invention relates to the use of an IFNγneutralizing molecule for preventing or treating T1 inflammatory lungdisease. More particularly, the present invention relates to the use ofan anti-IFNγ antibody for preventing or treating T1 inflammatory lungdisease, said antibody preferably being a monoclonal antibody.Furthermore, the present invention relates to the use of a human or ahumanized anti-IFNγ antibody for preventing or treating T1 inflammatorylung disease. More specifically, the present invention relates to theuse of the anti-IFNγ antibody D9D10, and more particularly a humanizedant-IFNγ antibody D9D10, for preventing or treating T1 inflammatory lungdisease.

Another aspect of the present invention relates to the use ofimmunogenic IFNγ for preventing or treating T1 inflammatory lungdisease, and in particular the use of human immunogenic IFNγ.

Accordingly, the present invention also relates to the prevention ortreatment of T1 inflammatory lung disease by immunization with apharmaceutical composition comprising immunogenic IFNγ proteins and/orIFNγ derived (poly)peptides. Several techniques to render IFNγimmunogenic are well known in the art and the present invention allowsfor all kinds of permutations of the original IFNγ sequence, and allkinds of modifications therein.

Another aspect of the invention relates to the use of the technology ofgenetic immunization, also known as “DNA vaccination”, for preventing ortreating T1 inflammatory lung disease.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the preferred methods and materials are nowdescribed. All publications mentioned hereunder are incorporated byreference. Unless mentioned otherwise, the techniques employed hereinare standard methodologies well known to one of ordinary skill in theart. The materials, methods and examples are only illustrative notlimiting.

The present invention provides compositions and methods for preventingand treating T1 inflammatory lung disease. Said lung diseases includebut are not limited to COPD (comprising emphysema, chronic bronchitis,and bronchiolitis), severe asthma, sarcoidosis, berylliosis and cysticfibrosis. The methods of the invention encompass the neutralizationand/or reduction and/or blockade of IFNγ bioactivity. The currentinvention thus relates to a method for preventing or treating T1inflammatory lung disease, said method comprising the neutralization ofIFNγ bioactivity. Several methods and/or compositions can be used inorder to achieve said effect and will be described hereunder.

Administration of IFNγ Neutralizing Molecules.

A first aspect of the invention is directed to the use of a moleculecapable of neutralizing and/or reducing and/or fully inhibiting thebioactivity of IFNγ for preventing or treating T1 inflammatory lungdisease. More specifically, the invention relates to the use of an IFNγneutralizing molecule for the manufacture of a medicament for preventingor treating a T1 inflammatory lung disease.

A “T1 inflammatory lung disease” is characterized by a Type 1 immuneresponse mediated by T helper-1 cells (CD4+) and T cytotoxic-1 cells(CD8+) and by predominant production of interferon gamma (IFNγ), tumornecrosis factor (IFNγ) and interleukin-2 (IL-2). T1 cytokines evokecell-mediated immunity characterized by prominent lung tissueinfiltration of macrophages, neutrophils and T-cells. Examples of T1inflammatory lung diseases include, but are not limited to COPD,emphysema, chronic bronchitis, bronchiolitis, severe asthma sarcoidosis,berylliosis, and cystic fibrosis.

As used herein, the term “molecule” encompasses, but is not limited to,an antibody and fragments thereof, a diabody, a triabody, a tetravalentantibody, a peptide, a low molecular weight non-peptide molecule (alsoreferred to as “small molecules”) and a (soluble) IFNγ receptor orfragments thereof, which specifically reduces and/or inhibits IFNγbioactivity. IFNγ of any species, including humans, is to be consideredin this respect.

As used herein, the term “antibody” refers to monoclonal antibodies,polyclonal antibodies, antibodies which are derived from a phagelibrary, humanized antibodies, synthetic antibodies, chimericantibodies, antibody fragments, single-chain Fv's, or constructsthereof. The term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notintended to be limited by the manner in which it is made. A monoclonalantibody typically displays a single binding affinity for a particularpolypeptide with which it immunoreacts. A monoclonal antibody to anepitope of the IFNγ antigen can be prepared by using a technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Kohler and Milstein (1975). Monoclonalantibodies can also be produced in various ways using techniques wellunderstood by those having ordinary skill in the art. Details of thesetechniques are described in “Antibodies: A Laboratory Manual”, Harlow etal.(ed), Cold Spring Harbor Publications, p. 726 (1988), or aredescribed by Campbell, A. M. (“Monoclonal Antibody Technology Techniquesin Biochemistry and Molecular Biology,” Elsevier Science Publishers,Amsterdam, The Netherlands (1984)) or by St Groth et al.( J. Immunol.Methods 35:1-21 (1980)). Monoclonal antibodies of any species, includinghumans, can be used in this invention. Accordingly, the antibodiesaccording to this embodiment may be human monoclonal antibodies. Suchhuman monoclonal antibodies may be prepared, for instance, by thegeneration of hybridomas, derived from immunised transgenic animals,containing large sections of the human immunoglobulin (Ig) gene loci inthe germline, integrated by the yeast artificial chromosomal (YAC)technology (Mendez et al., 1997). Also fragments derived from thesemonoclonal antibodies such as Fab, F(ab)′₂ and scFv (“single chainvariable fragment”), providing they have retained the original bindingproperties, form part of the present invention. The present inventionthus also relates to the use of an anti-IFNγ antibody for themanufacture of a medicament for preventing or treating T1 inflammatorylung disease, wherein said antibody is a monoclonal or polyclonalantibody, and more particularly a human monoclonal or polyclonalantibody.

As used herein, the term “humanized antibody” means that at least aportion of the framework regions of an immunoglobulin or engineeredantibody construct is derived from human immunoglobulin sequences. Itshould be clear that any method to humanize antibodies or antibodyconstructs, as for example by variable domain resurfacing as describedby Rogusim et al. (1994) or CDR grafting or reshaping as reviewed byHurle and Gross (1994), can be used.

As used herein, the term “chimeric antibody” refers to an engineeredantibody construct comprising variable domains of one species (such asmouse, rat, goat, sheep, cow, llama, or camel variable domains), whichmay be humanized or not, and constant domains of another species (suchas primate or human constant domains) (for review see Hurle and Gross(1994)). It should be clear that any method known in the art to developchimeric antibodies or antibody constructs can be used.

As used herein, the term “single chain Fv”, also termed scFv, refers toengineered antibodies prepared by isolating the binding domains (bothheavy and light chains) of a binding antibody, and supplying a linkingmoiety which permits preservation of the binding function. This forms,in essence, a radically abbreviated antibody, having only that part ofthe variable domain necessary for binding the antigen. Informationconcerning the generation, design and expression of recombinantantibodies can be found in Mayforth RD, “Designing Antibodies”, AcademicPress, San Diego (1993).

As used herein, the term “fragment” or “fragments” refers to F(ab),F(ab)′2, Fv, scFv and other fragments which retain the antigen bindingfunction and specificity of the parent antibody. The methods forproducing said fragments are well known to a person skilled in the artand can be found, for example, in Antibody Engineering, OxfordUniversity Press, Oxford (1995) (1996) and Methods in Molecular Biology,Humana Press, New Jersey (1995). In addition, any construct of anantibody or a fragment is also a subject of the current invention. Asused herein, the term “construct” relates to synthetic or recombinantmolecules, including but not limited to diabodies, triabodies,tetravalent antibodies, pepta- or hexabodies, and the like, that arederived from an anti-IFNγ antibody. The present invention thus relatesto the use of an IFNγ neutralizing molecule for the manufacture of amedicament for preventing or treating a T1 inflammatory lung disease,whereby said molecule is a construct derived from an anti-IFNγ antibody.

As used herein, the term “diabody” relates to two non-covalently-linkedscFv's, which then forming a so-called diabody, as described in detailby Holliger et al. (1993) and reviewed by Poljak (1994). It should beclear, that any method to generate diabodies, as for example describedby Holliger et al. (1993), Poljak (1994), and Zhu et al. (1996), can beused.

As used herein, the term “triabody” relates to trivalent constructscomprising 3 scFv's, and thus comprising 3 variable domains, asdescribed by Kortt et al. (1997) and Iliades et al. (1997). A method togenerate triabodies is described by Kortt et al. (1997). An example of atriabody is given in WO 99/09055 by Innogenetics N.V. It should also beclear that the scFv's, chimeric antibodies, diabodies and triabodiesdescribed above are not limited to comprise the variable domain of thesame antibody but may also comprise variable domains of other anti-IFNγantibodies which efficiently reduce or neutralize the bioactivity ofIFNγ. Furthermore, the diabodies or triabodies described above may alsocomprise two scFv's of different specificities. For example, the latterdiabodies may simultaneously neutralize IFNγ on the one hand and maytarget another molecule, such as TNF-α, IL-1, IL-2, B7.1 or CD80, B7.2or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6, complement factor,coagulation factor, fibrinolysis factor, tumour growth factor-beta(TGF-β), transferin receptor, insulin receptor and prostaglandin E2, orany other molecule, on the other hand.

As used herein the terms “IFNγ neutralizing molecule” or “IFNγneutralizing antibody” refer to a molecule and an antibody whichinhibits or blocks any bioactivity of IFNγ, respectively.

The term “bioactivity” or “biological activity” of IFNγ relates to theantiviral activity (Billiau, 1996), the induction of the expression ofMHC-class-II molecules by macrophages and other cell types (Steinman etal., 1980), the stimulation of the production of inflammatory mediatorssuch as TNF-α, IL-1 and NO (Lorsbach et al., 1993), the induction of theexpression of adhesion molecules such as ICAM-1 (Dustin et al., 1988)and of important costimulators such as the B7 molecules on professionalantigen presenting cells (Freedman et al., 1991), the induction ofmacrophages to become tumoricidal (Pace et al., 1983), the induction ofIg isotype switching (Snapper and Paul, 1987) or any other knownbioactivity of IFNγ. Billiau et al. (1996) describes pathological and/orclinical activity during diseases in which IFNγ is pathogenic It shouldbe noted that the molecules which neutralize IFNγ as described herein,neutralize at least one bioactivity but not necessarily allbioactivities of IFNγ. Tests to evaluate the effect of anti-IFNγmolecules or antibodies (i.e. IFNγ, neutralizing molecules or antibodiesresp.).on the bioactivity of IFNγ are available and well known to theskilled person. Examples of said tests are, but not limited to,“inhibition of MHCII-induction” and/or “inhibition of anti-viralactivity”. In the first mentioned test, the effect of IFNγ on theinduction of M19C class II expression on keratinocytes is examined. Forthis, primary keratinocytes are cultured with two concentrations ofIFN-γ (100 U/ml and 200 U/ml) for 24 and 48 hours. After culture, cellsare collected and the expression of MHC class II antigen on theactivated keratinocytes is measured by FACS-scan after staining (30minutes at 4° C) of the cells with a PE-labelled anti-MHC-class II mAb.In addition, the effect of an anti-IFNγmolecule on the IFNγ-inducedMHC-Class II expression on keratinocytes is examined. In thisexperiment, primary keratinocytes are cultured with IFNγ (100 U/ml) inthe presence or absence of different concentrations of anti-IFNγmolecules or antibodies for 48 hours. IFNγ is preincubated withanti-IFNγ molecules or antibodies for 1 hour at 37° C. before adding tothe keratinocytes. After culture, cells are collected and the expressionof MHC-Class II on these activated keratinocytes is measured. For this,keratinocytes are incubated (30 minutes at 4° C.) with a PE-labelledanti-MHC-ClassII mAb (Becton Dickinson), washed twice with PBS andfixed. The MHC-Class ll expression is further analysed on a FACS-scan.Analogous to the described test, the effect of IFNγ on the induction ofMHC-class II expression on B cells can be examined. Also otherexperiments known to those skilled in the art can be performed in orderto evaluate the neutralization capacity of anti-IFNγ molecules (incl.antibodies).

For the second test, whereby neutralization of the antiviral activity ofIFNγ is measured, serial dilutions of samples (anti-IFNγ molecules orantibodies) are prepared in microtiter plates. IFNγ is added to eachwell in a final concentration of 5 antiviral protection Units/ml, astested on A549 cells. The mixtures are incubated for 4 h at 37° C. and25000 A549 cells are added to each well. After an incubation period of24 at 37° C. a in CO₂ incubator, 25 μl of 8×10⁵PFU EMC virus/ml is addedto the cultures for at least 24 h. As soon as virus-infected controlcultures reach 100% cell destruction, a crystal violet staining isperformed in order to quantify surviving cells. The neutralizationcapacity of the anti-IFNγ molecules or antibodies can be defined forinstance, as the concentration of the molecule or antibody needed toneutralize 95% of the antiviral activity of 5U/ml IFNγ. Theneutralization potency of the anti-IFNγ molecules or antibodies is thendetermined. Further assays to evaluate the effect of anti-IFNγ molecules(incl. antibodies) on the bioactivity of IFNγ are described by e.g LewisJ. A. (1995), Kim Y. & Son K (1996) and by Maeger A. (2002).

The term “prevention” or “treatment” as used herein refers to either (i)the prevention of the disease of interest (prophylaxis), or (ii) thereduction or elimination of symptom exacerbations or the disease ofinterest (therapy), or (iii) any process, action, application, therapy,or the like, wherein a mammal, including a primate and more specificallya human being, is subject to medical aid with the object of improvingthe mammal's condition, directly or indirectly.

The present invention thus relates to a method for preventing ortreating T1 inflammatory lung disease comprising administering to apatient a pharmaceutically effective amount of an IFNγ neutralizingmolecule. More specifically, the present invention relates to a methodfor preventing or treating T1 inflammatory lung disease comprisingadministering to a patient a pharmaceutically effective amount of ananti-IFNγ antibody. According to a specific embodiment, the antibody isthe monoclonal antibody D9D10H3G5 produced by the hybridoma deposited onAug. 28, 2001, under the Accession No. DSM ACC2521, with theDSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1b, D-38124 Braunschweig, Germany. Said monoclonalantibody D9D10H3G5 will be further abbreviated throughout thespecification and the claims as D9D10. More specifically, the presentinvention relates to a method for preventing or treating T1 inflammatorylung disease comprising administering to a patient a pharmaceuticallyeffective amount of an anti-IFNγ antibody D9D10 or a fragment thereof.Furthermore, the present invention relates to the use of an anti-IFNγantibody for the manufacture of a medicament for preventing or treatingT1 inflammatory lung disease, said antibody preferably being amonoclonal antibody (e.g. D9D10 as described herein) or a humanizedmonoclonal antibody (e.g. Humanized D9D10 as described herein or asdescribed in W099109055 by Innogenetics N.V.).

Differently produced antibodies recognizing the same epitopes as theantibody D9D10, as well as antibodies immunologically competing with theantibody D9D10 for the binding on IFNγ are also part of the invention.Therefore, according to a further embodiment, the present inventionrelates to the use of an anti-IFNγ antibody or a fragment thereof forpreventing or treating T1 inflammatory lung disease, whereby saidantibody is characterized by its ability to immunologically compete withthe antibody D9D10 for the binding on IFNγ. As used herein, the term “tobind in an equivalent way” or “immunologically competing” means thatthese antibodies inhibit the binding of D9D10 to IFNγ and that theseantibodies neutralize the bioactivity of IFNγ. Preferred methods fordetermining antibody specificity and affinity by competitive inhibition,e.g. solid phase ELISA, can be found in Harlow et al. (1988), Colliganet al. (1992, 1993), Ausubel et al. (1987, 1992, 1993), and Muller R.(1993), and in Karlsson et al. (1991) and Malmqvist M. (1999).

As used herein, the tern “pharmaceutical composition” or “composition”or “medicament” refers to any composition comprising a molecule,including an antibody or fragment thereof, which specificallyneutralizes IFNγ, preferably in the presence of a pharmaceuticallyacceptable carrier or excipient. The present invention thus also relatesto the use of a pharmaceutical composition comprising at least one IFNγneutralizing molecule and an acceptable carrier for preventing ortreating T1 inflammatory lung disease. More preferably, said compositioncomprises the antibody D9D10 or a humanized D9D10 antibody and apharmaceutically acceptable carrier. Further, said compositionoptionally comprises other drugs or other antibodies, antibodyderivatives or constructs. With regard to COPD, emphysema, chronicbronchitis and chronic obstructive bronchiolitis, examples of such otherdrugs or other antibodies, antibody derivatives or constructs are, butare not limited to: bronchodilators, corticosteroids, Theophylline,antibiotics, Leukotriene B₄ (LT B₄) inhibitors, chemokine inhibitors,TNF-α inhibitors, anti-IL-8, antioxidants, iNOS inhibitors, neutrophilelastase inhibitors, cathepsin inhibitors, α₁-antitrypsin, secretoryleukoprotease inhibitors, elafin, PDE type IV inhibitors, NF-κBinhibitors, adhesion molecule blockers, IL-10, p38 MAP kinaseinhibitors, P13-kinase inhibitors and TNF-tip peptides as described inWO 00/09149; with regard to severe asthma: β2-agonists,anticholinergics, corticosteroids (e.g. prednisone); with regard tosarcoidosis: corticosteroids, cytotoxic agents, immunomodulators (e.g.chloroquine, hydroxychloroquine and TNF inhibitors such aspentoxifylline and thalidomide) and the antileprosy drugs clofazimineand minocycline; with regard to berylliosis: corticosteroids; withregard to cystic fibrosis: antimicrobial agents, corticosteroids andnon-steroidal anti-inflammatory drugs such as ibuprofen.

It should also be clear that any possible mixture of any IFNγneutralizing molecule, antibody or composition described in thespecification may be part of the above-indicated pharmaceuticalcomposition. The proportion and nature of said pharmaceuticalcompositions are determined by the solubility and chemical properties ofthe selected compound, the chosen route of administration, and standardpharmaceutical practice.

The IFNγ neutralizing molecule, antibody or a fragment thereof, and morepreferred the monoclonal antibody D9D10 or a humanized D9D10 antibody,or a fragment or construct thereof, may thus be administered ordelivered in the form of any suitable composition as described in thespecification by any suitable method of administration within theknowledge of one skilled in the art.

As used herein, the term “pharmaceutically acceptable carrier orexcipient”, whereby the term carrier and excipient are usedinterchangeably, refers to a diluent, adjuvant, or vehicle with whichthe therapeutic molecule is administered. It includes any and allsolvents, dispersion media, aqueous solutions, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, the usethereof in pharmaceutical compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions of theinvention A composition is said to be “pharmacologically acceptable” ifits administration can be tolerated by the recipient.

Immunization.

Another method to neutralize the bioactivity of IFNγ is by immunization.There is an increased focus on methods of instructing the recipient'sown immune system to generate endogenous antibodies of the appropriatespecificity by means of immunization. However, mammals do not generallyhave high-titre antibodies against self-proteins in serum because ofhomeostatic tolerance mechanisms that prevent their formation(autotolerance).

It has been shown (by Dalum I. et al., 1996) that potentiallyself-reactive B lymphocytes recognizing self-proteins arephysiologically present in normal individuals. However, in order forthese B lymphocytes to be induced to actually produce antibodiesreactive with the relevant self-proteins, assistance is needed fromcytokine producing T helper lymphocytes (Th-cells or Th-lymphocytes).Normally this help is not provided because T lymphocytes in general donot recognize T-cell epitopes derived from self-proteins when presentedby antigen presenting cells (APCs) such as dendritic cells, macrophagesand B cells. However, by providing an element of “foreignness” in aself-protein, T-cells recognizing the foreign element are activated uponrecognizing the foreign epitope on an APC (such as, initially, amononuclear cell). Polyclonal B lymphocytes (which are also specialisedAPCs) capable of recognizing self-epitopes on the modified self-protein,also internalize the antigen and subsequently present the foreign T-cellepitope(s) thereof, and the activated T lymphocytes subsequently providecytokine help to these self-reactive polygonal B lymphocytes. Since theantibodies produced by these polyclonal B lymphocytes are reactive withdifferent epitopes on the modified polypeptide, including those whichare also present in the native polypeptide, an antibody cross-reactivewith the non-modified self-protein is induce. In conclusion, the Tlymphocytes can be led to act as if the population of polyclonal Blymphocytes have recognized an entirely foreign antigen, whereas in factonly the added or inserted epitope(s) is/are foreign to the host. Inthis way, antibodies capable of cross-renting with non-modifiedself-antigens are induced.

Different kinds of immunization approaches that are able to break B-celltolerance and circumvent antibody tolerance mechanisms without inducingauto-antibody-mediated pathology and toxicology are well known by theskilled person and are outlined hereunder.

Accordingly, the present invention relates to the prevention ortreatment of T1 inflammatory lung disease by immunization with apharmaceutical composition comprising immunogenic IFNγ proteins and/orIFNγ derived (poly)peptides. These IFNγ-related compounds are capable ofraising auto-antibodies and/or T cells when administered in vivo. Theautoantibodies are able to neutralize and inhibit the biologicalactivities of endogenously produced IFNγ. The present invention thusrelates to the use of immunogenic IFNγ or IFNγ derived (poly)peptidesfor the manufacture of a medicament for preventing or treating a T1inflammatory lung disease.

“Immunization” means that a substance or composition of matter exhibitsor induces an immune response resulting in endogenous antibodyproduction concomitant with or without T cell help. Endogeneous antibodyproduction against IFNγ can be measured using standard techniques, e.g.by ELISA.

The term “immunogen” is intended to denote a substance which is capableof inducing an immune response in a certain mammals, including primatesand more specifically humans. It will therefore be understood thatautologous IFNγ is not an immunogen in the autologous host undernon-pathogenic conditions. It is necessary to use either a strongadjuvant and/or to co-present foreign T helper epitopes with theautologous INFγ in order to mount an immune response against autologousIFNγ and in such a case the “immunogen” is the composition of matterwhich is capable of breaking autotolerance. Other immunogens describedin the current invention include but are not limited to modified IFNγ,non-self IFNγ (i.e. IFNγ from another species) and antigen presentingcells loaded with IFNγ. The term “immunogenic IFNγ” thus specificallyrelates to:

-   -   modified IFNγ and/or,    -   non-self IFNγ derived from another species (mammalian or other)        administered with or without an adjuvant and/or,    -   autologous IFNγ co-presented or in combination with an adjuvant        and/or,    -   autologous antigen presenting cells, such as dendritic cells,        loaded with IFNγ.

The expressions “IFNγ”, “IFNγ protein” and “IFNγ polypeptide”, which areused interchangeably, refer to a family of polypeptide molecules thatinclude human IFNγ from natural sources, synthetically produced invitro, or obtained by genetic manipulation including methods ofrecombinant DNA technology. The amino acid sequence variants preferablyshare at least about 65% sequence homology, more preferably at leastabout 75% s c homology, even more preferably at least about 85% sequencehomology, most preferably at least abut 90% sequence homology with anydomain, and preferably with the receptor binding domain(s) of the nativehuman IFNγ amino acid sequence. Several databases and tools fordetermining amino acid homology are known by the person skilled in theart, e.g. BLAST®, and are described by Gish W. & States D. J. (1993) andin “Bioinformatics: sequence and genome analysis”, Mount (ed.), ColdSpring Harbor Laboratory Press (2001). The definition specificallycovers variously glycosylated and unglycosylated forms of native humanIFNγ and of its amino acid sequence variants.

A “modified IFNγ” is an protein or IFNγ derived (poly)peptide which hasbeen subjected to changes in its primary structure. Such a change cane.g. be in the form of fusion or conjugation of an IFNγ polypeptide to asuitable fusion partner (i.e. a change in primary structure exclusivelyinvolving C-and/or N-terminal additions of amino acid residues) and/orit can be in the form of insertions and/or deletions and/orsubstitutions in the amino acid sequence of IFNγ. It should also benoted that the IFNγ protein can become immunogenic due to certainmodifications resulting from the recombinant production process,isolation, handling, or storage of the protein. According to a preferredembodiment of the present invention the IFNγ protein is renderedimmunogenic by its modification.

One technique involves chemically crosslinking of the IFNγ self-protein(or (poly)peptide(s) derived from it) to a highly immunogenic non-selfcarrier protein such as, but not limited to, keyhole limpet haemoncyanin(Kim, 2001), ovalbumin (Richard, 2000), tetanus toxoid, detoxifieddifteria or cholera toxin (Talwar, 1999), bacterial outer membraneproteins (Gonzalez, 2000), E.coli enterotoxin (Lowenadler, 1994), heatshock proteins (Udono, 1993), and viral-like particles (Chackerian,2002; Storni, 2002). The carrier protein contains several differentforeign T-cell epitopes needed to trigger the activation of T-cellswhich in their turn provide help to B-cells that produce antibodiesspecifically directed against the conjugated IFNγ self-antigen. Insteadof whole carrier proteins, the chemical cross-linking of foreign MHCClass II restricted T-cell epitopes may also be efficient for theinduction of auto-immune responses against IFNγ (Sad, 1992). The currentinvention thus relates to the use of immunogenic IFNγ for themanufacture, of a medicament for preventing or treating a T1inflammatory lung disease, whereby the IFNγ protein is modified toimmunogenic IFNγ by crosslinking IFNγ to an immunogenic non-pelf carrierprotein. Furthermore, the current invention also relates to the use ofimmunogenic IFNγ for the manufacturing of a medicament for preventing ortreating a T1 inflammatory lung disease, whereby the IFNγ protein ismodified to immunogenic IFNγ by chemical cross-linking of one or moreforeign MHC Class II restricted T-cell epitopes.

A variant on the carrier protein technique involves the construction ofa gene encoding a fusion protein comprising both carrier protein orforeign T-cell epitope(s) and the IFNγ self-protein or B-cell epitope(s)derived therefrom. The fusion protein may be pressed in a suitable hostcell in vitro, the gene product purified and then delivered as aconventional pharmaceutical composition co-presented with or withoutadjuvant. The current invention thus also relates to the use of a fusionprotein for the manufacture of a medicament for preventing or treating aT1 inflammatory lung disease, whereby the fusion protein comprises acarrier protein or at least one foreign T-cell epitope and the IFNγself-protein or B-cell epitope(s) derived therefrom. Alternatively, thefusion gene may be administered directly as part of a nucleic acidvaccine.

The foreign T-cell epitope(s) can not only be conjugated to the amino-or carboxy-terminal end of the human IFNγ self-protein but canalternatively be inserted into the human IFNγ protein either at randomor at sites predicted not to interfere with the general conformationalstructure. As a result, T-cell help arises either from this epitope(s)or from junctional sequences.

A more refined approach has been described by Dalum and colleagueswherein a small part of target molecule is substituted by a single MHCClass II restricted T cell epitope (Dalum I., 1999). The same approachcan be used to introduce multiple epitopes by substitution. Thesubstituted epitope is supposed not to interfere with the folding of theprotein resulting in the adoption of a fully native conformationidentical to the one found in the native endogenous self-proteinTherefore, it is highly probable that antibodies recognizing theimmunogen will also recognize and at best neutralize the endogenousself-protein.

One preferred version of this embodiment is the technique described inWO 95/05849 by Elsner Henrik et al., which discloses a method fordown-regulating self-proteins by immunizing with analogues of theself-proteins wherein a number of amino acid sequence(s) has beensubstituted with a corresponding number of amino acid sequence(s) whicheach comprise a foreign immunodominant T-cell epitope, while at the sametime maintaining the overall tertiary structure of the self-protein inthe analogue. For the purposes of the present invention it is, however,sufficient if the modification (be it an amino acid insertion, addition,deletion or substitution) gives rise to a foreign T-cell epitope and atthe same time preserves a substantial number of the B-cell epitopes inIFNγ. The present invention thus relates to the use of immunogenic IFNγfor the manufacture of a medicament for preventing or treating a T1inflammatory lung disease, wherein the IFNγ protein is modified toimmunogenic IFNγ by introducing at least one foreign T-cell epitope intoIFNγ by insertion, substitution, addition and/or conjugation. Saidforeign T-cell epitope can be any natural or synthetic T-cell epitope asdescribed herein.

A “foreign T-cell epitope” is a peptide which is able to bind to an MHCmolecule and stimulates T-cells in an animal species. Preferred foreignepitopes are “promiscuous” epitopes, i.e. epitopes which bind to asubstantial fraction of polymorphic MHC class II molecules in an animalspecies. A term which is often used interchangeably in the art is theterm “universal T-cell epitopes” for this kind of epitope.

A number of naturally occuring “promiscuous” T-cell epitopes exist whichare active in a large proportion of individuals of an animal species,including human, and these are preferably introduced in the IFNγcomposition thereby reducing the need for a very large number ofdifferent analogues in the same composition.

The promiscuous epitope can according to the invention be a naturallyoccurring human T-cell epitope such as epitopes from tetanus toxoid (e.g. the P2 and P30 epitopes in WO 00/20027), diphtheria toxoid, influenzavirus hemagluttinin (HA), and P. falciparum CS antigen.

Over the years a number of other promiscuous T-cell epitopes have beenidentified. in particular, peptides capable of binding a largeproportion of HLA-DR molecules encoded by the different HLA-DR alleleshave been identified and these are all possible T-cell epitopes to beintroduced in analogues used according to the present invention.Additional epitopes are discussed in the following references: Kilgus etal., 1991; Contreras et al., 1998; Doolan et al., 2000, Launois et al.,1994; Mustafa et al., 2000; Fernando et al., 1995; Gaudebout et al.,1997; Friedl-Hayek et al., 1999; Kobayashi et al., 2000. All epitopeslisted in these references are relevant as candidate epitopes to be usedin the present invention, as are epitopes which share common motifs withthese.

Alternatively, the epitope can be any synthetic or artificial T-cellepitope which is capable of binding a large proportion of haplotypes. Inthis context the pan DR epitope peptides (“PADRE”) described in WO95/07707 and in the corresponding paper by Alexander J. et al. (1994)are interesting candidates for epitopes to be used according to thepresent invention.

Another mechanism provides for the generation of a multiplicity ofpotential T-cell epitopes, yet simultaneously retains the targetmolecule in a conformation close to the native form (Ciapponi,1997).These properties can be achieved by rendering one or several mutationsin a self-protein to produce a sequence at those points which can befound in an analogous protein from a second mammalian species. Mutationscan be applied to consecutive amino acids or locally dispersed aminoacids.

The current invention relates to the use of immunogenic IFNγ for themanufacture of a medicament for preventing or treating a T1 inflammatorylung disease, whereby the IFNγ protein is modified to immunogenic IFNγby rendering one or several mutations in the IFNγ self-protein. Suchmutations may in effect produce a sequence at those points which can befound in an analogous protein from a second mammalian species.Alternatively, B-cell epitopes of IFNγ from a first ma an species may begrafted (e.g. by insertion or substitution) into the framework of aprotein from a second mammalian species such that the modified proteinis able to raise in the first species an immunogenic response directedto the natural IFNγ protein from which the B-cell epitopes are derived.A specific embodiment of the current invention thus relates to the useof immunogenic IFNγ for the manufacture of a medicament for preventingor treating a T1 inflammatory lung disease, whereby a protein from asecond mammalian species is modified to an immunogen by B-cell epitopesfrom an IFNγ protein from a first mammalian species into the frame workof protein from the second mammalian species. Preferably, the firstmammalian species is human.

Auto-antibodies in humans can also be induced by immunization with anon-modified analogous protein from a second mammalian species(Vernersson, 2002). Antibodies directed against the analogous proteinare suspected to cross-react with the human self-protein resulting inthe neutralization of its biological activity. The invention thus alsorelates to the use of immunogenic IFNγ for the manufacture of amedicament for preventing or treating a T1 inflammatory lung disease,whereby the immunogenic IFNγ is a non-self IFNγ derived from a secondmammalian species.

Anti-human IFNγ auto-antibodies in humans can also be achieved byimmunization with autologous antigen presenting cells (APCs) ex vivoloaded with human IFNγ (Li, 2002). The invention thus also relates tothe use of at least one autologous antigen presenting cell loaded withIFNγ for the manufacture of a medicament for preventing or treating a T1inflammatory lung disease. More particularly, the invention relates tothe use of at least one autologous dendritic cell loaded with IFNγ forthe manufacture of a medicament for preventing or treating a T1inflammatory lung disease

Thus, given the general functional restraints on the immunogenicity ofthe constructs, the invention allows for all kinds of permutations ofthe origin IFNγ sequence, and all kinds of modifications therein.

Some of the IFNγ proteins of the pharmaceutical composition aresufficiently immunogenic, but for some the immune response will beenhanced if the composition further comprises an adjuvant substance. Itis especially preferred to use an adjuvant which can be demonstrated tofacilitate breaking of the autotolerance to autoantigens. Variousmethods of achieving adjuvant effect for the composition are known.General principles and methods are detailed in “The Theory and PracticalApplication of Adjuvants”, 1995, Duncan B. S. Stewart-Tull (ed.), JohnWiley & Sons Ltd., and also in “Vaccines : New Generation ImmunologicalAdjuvants”, 1995, Gregoriadis G et al. (eds.), Plenum Press. New York.

Preferred adjuvants facilitate uptake of the molecules by APCs, such asdendritic cells, and activate these. Non-limiting examples are selectedfrom the group consisting of an immune targeting adjuvant; an immunemodulating adjuvant such as a toxin, a cytokine, and a mycobacterialderivative; an oil formulation ; a polymer, a micelle forming adjuvant;a saponin; an immunostimulating complex matrix (ISCOM matrix); aparticle; DDA aluminium adjuvants; DNA adjuvants; y-inulin; and anencapsulating adjuvant Details relating to composition and use ofimmunostimulating complexes can e.g. be found in the above-mentionedtext-books dealing with adjuvants, but also Morein B. et al., 1995, aswell as Barr I. G. and Mitchell G. F., 1996, provide useful instructionsfor the preparation of complete immunostimulating complexes. The currentinvention thus also relates to the use of a pharmaceutical compositioncomprising immunogenic IFNγ and an adjuvant for treating T1 inflammatorylung disease. More specifically, the current invention relates to theuse of a pharmaceutical composition comprising autologous IFNγ and anadjuvant for treating T1 inflammatory lung disease.

Another approach of immunization includes the technology of nucleic acidimmunization, also known as “genetic immunization”, “gene immunization”and “DNA vaccination”. The present invention thus also relates to theuse of a DNA vaccine for preventing or treating T1 inflammatory lungdisease. In this embodiment, the introduced nucleic acid encoding themodified IFNγ protein as described herein is preferably DNA which can bein the form of naked DNA, DNA formulated with charged or unchargedlipids, DNA formulated in liposomes, emulsified DNA, DNA included in aviral vector, DNA formulated with a transfection-facilitating protein orpolypeptide, DNA formulated with a targeting protein or polypeptide, DNAformulated with calcium precipitating agents, DNA coupled to an inertcarrier molecule, and DNA formulated with an adjuvant. In this contextit is noted that practically all considerations pertaining to the use ofadjuvants in traditional vaccine formulation apply to the formulation ofDNA vaccines. Hence, all disclosures herein which relate to use ofadjuvants in the context of protein or (poly)peptide basedpharmaceutical compositions apply mutatis mutandis to their use innucleic acid vaccination technology. The same holds true for otherconsiderations relating to formulation and mode and route ofadministration and, hence, also these considerations discussed herein inconnection with a traditional pharmaceutical composition apply mutatismutandis to their use in nucleic acid vaccination technology.

Accordingly, the present invention also relates to a method ofpreventing or treating Tγ inflammatory lung disease by immunization witha pharmaceutical composition comprising a nucleic acid sequence encodinga immunogenic IFNγ protein and/or IFNγ-derived (poly)peptide. Morespecifically, the present invention relates to the use of a nucleic acidsequence encoding an immunogenic IFNγ protein and/or IFNγ derived(poly)peptide for the manufacture of a medicament for preventing ortreating a T1 inflammatory lung disease.

Under normal circumstances, the nucleic acid of the vaccine isintroduced in the form of a vector wherein expression is under controlof a viral promoter.

Therefore, also provided are an expression vector which comprises apolynucleotide of the herein described proteins or peptides and which iscapable of expressing the respective proteins or peptides, a host cellcomprising the expression vector and a method of producing and purifyingherein described proteins or peptides, pharmaceutical compositionscomprising the herein described proteins or peptides and apharmaceutically acceptable carrier and/or adjuvants.

Detailed disclosures relating to the formulation and use of nucleic acidvaccines are available, e.g. by Donnelly J. J. et al., 1997 and 1997a.

Administration

The molecule, protein, composition or agent of the current invention maybe administered in any manner which is medically acceptable. Inaddition, it can at any time be administered together, simultaneously orsequentially, with another separate substance, molecule, antibody,composition, or a pharmaceutically and immunologically acceptablecarrier and/or vehicle and/or an adjuvant.

Any of the conventional methods for administration of a pharmaceuticalcomposition are applicable e.g parenterally, orally or by eithersubcutaneous, intradermal, subdermal, intravenous, intaarterial orintramuscular injection. Other modes of administration includesuppositories and, in some cases, buccal, sublinqual, intraperitoneal,intravaginal, anal, and intracranial applications. Depending on thespecific circumstances, local or systemic administration may bedesirable.

One skilled in the art of preparing formulations can readily select theproper form and mode of administration depending upon the particularcharacteristics of the molecule, protein, composition or agent selected,the disease state to be treated, the stage of the disease, and otherrelevant circumstance.

According to the specific case, the “pharmaceutically effective amount”or “amount effective” is one that is sufficient to produce the desiredeffect. This can be monitored using several end-points known to thoseskilled in the art such as, but not limited to, mortality, morbidity,and the like. According to the specific case, the pharmaceuticallyeffective amount should be determined as being the amount sufficient tocure the recipient in need of treatment, to prevent or at least topartially reduce or halt the disease or injury and its complications.The term “recipient” is intended to include living organisms, e.g.mammals, primates, and more specifically humans. Amounts effective forsuch use will depend on the severity of the disease and the generalstate of the recipient's health As such, dosage of the administeredmolecule, protein, composition or agent will vary depending upon suchfactors as the recipient's age, weight, height, sex, general medicalcondition, previous medical history, concurrent treatment with otherpharmaceuticals, etc. Administration can be as a single dose or repeateddoses one or more times after a specific period. When administering byinjection, the administration may be by continuous injection, or bysingle or multiple boluses. Typically, such compositions are prepared asinjectables either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in liquid prior to injection mayalso be prepared. The preparation may also be emulsified.

The active ingredient of the pharmaceutical composition is often mixedwith excipients or carriers which are pharmaceutically acceptable andcompatible with the active ingredient. Such excipients are inherentlynontoxic and nontherapeutic. Examples of such excipients are water,saline, glycerol, ethanol, Ringer's solution, dextrose solution andHank's solution, and/or combinations thereof. Nonaqueous excipients suchas fixed oils and ethyl oleate may also be used. A preferred excipientis 5% dextrose in saline. The excipient may contain minor amounts ofadditives such as substances that enhance isotonicity and chemicalstability, including buffers and preservatives. In addition, if desired,the composition may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, or adjuvantswhich enhance the effectiveness of the composition. Examples ofadjuvants are sterile diluents such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose.

LEGEND TO THE FIGURE

FIG. 1: TNF-alfa levels were measured in the serum of the animals after6 weeks of smoking. Sera from 3 placebo-treated animals and from 3animals from the anti-IFNgamma treated group were tested.TNF-alfa wasmeasured using an in house developed ELISA. The detection limit is 4pg/ml. As shown in this figure, treatment of the smoking animals withanti-IFNgamma inhibits the TNF-alfa production.

EXAMPLES Example 1:

Measurement of IFNγ in Lungs of Patients With T1 Inflammation

Immunological inflammation may be of a Type 1 with predominance ofinterleukin 2 (IL2) and interferon gamma (IFNγ) production or a Type 2characterized by predominant IL-4 and IL-5 production. Presence of IFNγin the lungs of patients with T1 inflammatory lung diseases can bemeasured in different ways as outlined hereunder.

One method is the measurement of cytokine levels in spontaneouslyproduced and/or induced sputum. Sputum is defined as expectorations offluid, cells and solutes that are present in the lining fluid of theupper bronchial tree. Spontaneous sputum can be obtained in a simple andnon-invasive way whilst induced sputum is induced by exposure ofindividuals to a nebulised saline solution (Out et al. 2001). Proteaseinhibitors can be added shortly after isolation.

The gel and sol phase in sputum can be separated from each other bymeans of ultacentrifugation (50.000 g; 4° C.). Otherwise, mucus can behomogenized by treatment with dithiotreitol (DTT) (Karpati et al.,2000). Additional treatment by DNAse to degrade any DNA present ortreatment with trypsin-EDTA may be necessary. Further low speedcentrifugation (300 g) of homogenised sputum allows the separation ofsupernatant from the cellular compartment. Supernatant can be used assuch or concentrated and eventually stored at −70° C. until assessmentof cytokine levels.

BAL fluid is obtained by standard lavage protocol consisting of infusing20 ml aliquots of sterile saline solution through an aspiration portfollowed by lavage collection through same. This procedure is repeated 5times.

Sputum and/or BAL is collected from severe COPD patients (FEV1<30%predicted according to the GOLD criteria), moderate COPD patients (30%≦FEV1<80% predicted according to the GOLD criteria), pack-year matchedhealthy smokers (normal FEV1 and no chronic symptoms of cough or sputumproduction) and control non-smokers. Patients are well characterizedage, gender, pack-years smoking history, lung function test, acuteexacerbations, inflammation.

Fluid phase of sputum or BAL is evaluated for presence of IFNγ by meansof the Enzyme Linked Immunosorbent Assay (ELISA) method. A proteolyticenvironment like sputum and the treatment of sputum with reducing agents(DTT) may affect antigenic determinants as well as the characteristicsof the antibodies and thus the performance of the immunoassays.Therefore, a particular assay has to be validated for its application insputum. Commercially available kits from Medgenix or R&D Systems orAmersham Biosciences can be used to determine IFNγ immunoreactivities(Keatings, 2002).

Cellular fractions can be assessed for the presence of relativeproportions of different cell types by Wright stain on cytospin slides.Cellular fractions can further be analysed for IFNγ production, by,immunohistochemistry on cytospin slides or RT-PCR on total cell RNA orby FACS analysis of intracelluar cytokines.

The leakage of plasma proteins (e.g. albumin, fibrinogen) from the bloodinto the airspace nay be considered as an overall marker forinflammation.

Presence of IFNγ can also be assessed in biopsies of smokers,non-smokers or ex-smokers with mild COPD or in lung fragments obtainedby lobectomie of severe COPD patients. IFNγ can be evaluated byimmunohistochemistry or RT-PCR.

Example 2:

Effect of Blocking Ifn-gamma, by Anti-mouse Ifn-gamma Mab, on CigaretteSmoke Induced Emphysema in Mice

Introduction

Chronic Obstructive Pulmonary Disease (COPD) is characterized by theprogressive development of a not fully reversible airflow limitation(Pauwels et al., 2001). The airflow limitation is due to a variablemixture of respiratory bronchiolitis and emphysema. A major risk factorfor COPD is cigarette smoking. Inflammation of the airways and the lungsis thought to play a major role in the pathogenesis of COPD. Humanstudies clearly illustrate a smoking-induced inflammatory response,comprised of neutrophils, macrophages, dendritic cells, eosinophils,CD4⁺ and CD8⁺ T-lymphocytes in smokers' airways and lung parenchyma(Retamales et al., 2001; Casolaro et al., 1988). The exact role ofindividual inflammatory cells and mediators is unknown at the moment.

Wang et al. (2000) demonstrated that interferon gamma (IFN-gamma) causesemphysema with alveolar enlargement, enhanced lung volumes, enhancedpulmonary compliance, and macrophage- and neutropbil-rich inflammationwhen inducibly targeted, in a transgenic fashion, to the adult murinelung. Prominent protease and antiprotease aerations were also noted inthese mice. They included the induction and activation of matrixmetalloproteinase (MMP)-12 and cathepsins B, H, D, S, and L, theelaboration of MB-9, and the selective inhibition of secretory leukocyteproteinase inhibitor.

The aim of the current study is primary to investigate the effect ofblocking IFN-gamma on the development of emphysema in mice exposed forsix months to cigarette smoke. A secondary objective is to study theeffect of anti-mouse IFN-gamma mAb on the cigarette smoke inducedpulmonary inflammation.

Design of the Study

Three groups of 15 mice are followed for 24 weeks.

Group I is exposed to room air and treated twice a week (Monday andThursday) intraperitoneally with vehicle.

Group II is exposed to cigarette smoke 5 days a week and treated twice aweek (Monday and Thursday) intraperitoneally with vehicle.

Group III is exposed to cigarette smoke 5 days a week and treated twicea week (Monday and Thursday) intraperitoneally with 100 μg anti-mouseIFN-gamma mAb, F3.

Blood samples (300-400μl) are taken weekly Thursday) from the ocularsinus. Serum is prepared from each sample. Serum concentrations ofanti-IFN-gamma mAb F3. IFN-gamma IL-6. TNF alfa IL-8. IL-16, differentchemokines and other parameters, are analyzed using ELISA technology. NOwas measured using Colormetric Nitric Oxide detection kit.

Immediately following the last exposure, animals are sacrificed andemphysema and lung inflammation are quantified using the followingparameters:

Emphysema: Mean Linear Intercept, Mean Alveolar Surface, Mean number ofalveolar wall breaks/alveolar space.

Inflammation:

BAL fluid: total number of cells, differential cell count formacrophages/monocytes, neutrophils, eosinophils and lymphocytes.

Lung tissue (single cell suspension): total number of cells,macrophages, dendritic cells, lymphocytes (CD3+), CD4+lymphocytes,CD8+lymphocytes, activated CD4+lymphocytes, activated CD8+lymphocytes.

Methods

Animals

Male C57B1/6 mice, 10 wks old (at the start of the experiment i.e.pre-bleeding), are purchased from Harlan (Zeist, The Netherlands). Themice are kept in standard animal research facilities and receive foodand water ad libitum. Mice are kept in groups of 6. The local EthicsCommittee approved all in vivo manipulations.

Experimental Design

Groups of 5 mice are exposed to the tobacco smoke of 5 cigarettes(Reference Cigarette 1R3, University of Kentucky, Lexington) perexposure. There ate 4 exposures a day with a 30 minutes smoke-freeinterval between each exposure. The animals smoke 5 days a week for upto 24 weeks. The control group is exposed to air. At week 24, mice aresacrificed and inflammatory parameters are examined in bronchoalveolarlavage (BAL) fluid and lung single cell suspension. Besides,histological evaluation of lung parenchyma is performed.

Tobacco Smoke Chamber

Mice are exposed to cigarette smoke, with the use of a smoking apparatus(S. Shapiro, Washington University Medical Center, USA) with the chamberadapted for a group of 6 mice (chamber volume of 7500 cm³). An optimalsmoke:air ratio of 1:12 is obtained by injecting smoke and pressurizedair at a flow rate of 200 ml/min and 2.4 L/min respectively.

Bronchoalveolar Lavage

Animals are sacrificed after i.p. injection of an overdose ofpentobarbital (Sanofi, Libourne, France) and the trachea is surgicallyexposed and cannulated to perform bronchoalveolar lavage. 1 ml of Hank'sbalanced salt solution (HBSS) (GIBCO BRL), free of ionized calcium andmagnesium, and supplemented with 0.05 mM EDTA (Sigma) is instilled 3times via the tracheal cannula and recovered by gentle manualaspiration. The recovered bronchoalveolar lavage fluid (BAL) iscentrifuged, the cell pellet is washed twice and finally resuspended in1 ml of HBSS. A total cell count is performed in a Bürcker chamber andthe differential cell counts (on at least 400 cells) are performed oncytocentrifuged preparations after May-Grünwald-Giemsa staining. Flowcytometric analysis of BAL-cells is also performed (see below).

The supernatant of the BAL is stored at −80° C. for measurement ofIFN-gamma

Buffers and Media for Preparation of Single Cell Suspensions andImmunofluorescent Labeling

Digestion medium consists of RPMI 1640 supplemented with 2 mML-glutamine, 10 μg/ml streptomycine, 100 U/ml penicilliin, 5% FCS,0.001% β-mercaptoethanol (all from GIBCO BRL), 1 mg/ml collagenase type2 (Worthington Biochemical Corp.), and 0.02 mg/ml DNase I (grade II frombovine pancreas; Boehringer). FACS-EDTA buffer contains PBS (GIBCO BRL)without Ca²⁺ or Mg²⁺, 0.1% azide, 5% EDTA-treated FCS, and 5 mM EDTA.EDTA-treated FCS is prepared by passing FCS though a 0.2 μm filter andmixing 1 ml of a 0.1 M disodium EDTA solution with 10 ml of filteredFCS.

Preparation of Lung Single Cell Suspensions

Following BAL the pulmonary and systemic circulation is perfused withsaline containing 5 mM EDTA to remove the intravascular pool of cells.One lung is used for histology, the other is used for the preparation ofa cell suspension. The lung is thoroughly minced using iridectomyscissors and incubated for 30 min in digestion medium in a humidifiedincubator at 37 ° C. and 5% CO₂. Organ fragments are resuspended, freshdigestion medium is added, and incubation is extended for another 15min. After a final resuspension, very few organ debris are left. Samplesare centrifuged and resuspended in calcium and magnesium-free PBScontaining 10 mM EDTA at room temperature. Finally, the cells aresubjected to RBC lysis, washed in FACS-EDTA, passed through a 50 μm cellstainer, and kept on ice until labeling. Cell counting is performed witha Z2 Beckman-Coulter particle counter (Beckman-Coulter).

Labeling of BAL-cells and Lung Single Cell Suspensions for FlowCytometry

Cells are pre-incubated with Fc-receptor blocking antibody(anti-CD16/CD32, clone 2.4G2) to reduce non-specific binding. Monoclonalantibodies used to identify mouse DC populations are: biotinylatedanti-CD11c (N418) and PE-conjugated anti-IA^(b) (AF6-120.1), followed bystreptavidine-allophycocyanine (Sav-APC) (all from BD PharMingen).Isotype controls are PE-conjungated rat IgG_(2a,K), rat IgG_(2b) andarmenian hamster IgG_(2,K). The following antibodies are used to stainmouse T-cell subpopulations: FITC-conjugated anti-CD4 (L3T4),FITC-conjugated anti-CD8 (Ly-2), and biotinylated anti-CD3 (145-2C11)monoclonal antibodies. The additional marker used for activation isanti-CD69 (H1.2F3). Biotinylated anti-CD3 is revealed by incubation withSav-APC (all from BD Pharmingen). Monocytes/macrophages will beidentified using FSC/SSC and CD11c staining.

As a last step before analysis cells are incubated with7-amino-actinomycin (7-AAD or viaprobe, BD Pharmingen) for dead cellexclusion. All labeling reactions are performed on ice in FACS-EDTAbuffer.

Flow Cytometry data acquisition is performed on a dual-laser FACSVantage™ flow cytometer running CELLQuest™ software (Becton Dickinson).FlowJo software (www.Treestar.com) is used for data analysis.

Histology

After clamping the main bronchus of the lung excised for the preparationof a cell suspension, fixative (4% paraformaldehyde in PBS) is gentlyinfused through the tracheal cannula by a continuous-release pump underpressure and volume controlled conditions (12 ml/hour, 3 psi, 10min/lung). The lung is resected and fixed for an additional 4 h. Afterroutine paraffin embedding, 3 μm sections are stained with hematoxylinand eosin (H&E) (Klinipath) and examined by light microscopy forhistological changes.

Quantification of emphysema is performed in a blinded fashion using aZeiss KS400 Image Analyzer system running a custom-made morphometryprogram The mean linear intercept (L_(m)) is measured for each mousefrom 10 random fields by means of a 100×100 μm grid [15]. The meanalveolar surface (A_(m)) is also measured in 10 random fields per mouse.The number of alveolar wall breaks is counted per field and divided bythe number of alveolar spaces in that field. The mean number ofbreaks/alveolar space is calculated from 10 random fields per mouse.

Results:

1. Effect of Anti-IFN-gammma mAb Treatment on Circulating TNF-alfaLevels Induced in Smoking Animals

Proinflammatory cytokines, particularly IL-1beta and TNF, may amplifythe inflammatory response in COPD and be linked to disease severity.TNF-alfa is present in high concentrations in the sputum of COPDpatients. Serum concentration of TNF-alfa are increased in weight-losingCOPD patients, suggesting that it may play a role in the cachexia ofsevere COPD. TNF-alfa inhibits the expression of skeletal muscleproteins via activation of NF-kB. This suggests that inhibitors ofTNF-alfa might be useful in reversing the skeletal wasting seen in COPDas well as reducing the airway inflammatory response (Barnes, 2003;Oudijk, 2003)

In our experiment, inflammation in the smoking animals is evidenced byincreased levels of TNF-alfa in the circulation of the smoking mice, ascompared to the non-smoking animals. TNF alfa is detectable in thesmoking animals after 3 to 4 weeks of smoking. TNF-alfa levels aredecreased by treatment of the animals with anti-IFNgamma mAb, as shownin FIG. 1.

Our results demonstrate that blocking IFNgamma has an effect on thesystemic levels of TNF-alfa. Thus, these data demonstrate that blockingIFN-gamma has an effect on the inflammatory response induced by smokingin these animals.

REFERENCES

-   Aaron S D, Angel J B, Lunau M, et al. 2001. Granulocyte inflammatory    markers and airway infection during acute exacerbation of chronic    obstructive pulmonary disease. Am. J. Resp. Crit. Care Med    163:349-55-   Alexander J., Sidney J., Southwood S., Ruppert J., Oseroff C.,    Maewal A., Snoke K., Serra H M., Kubo R T., Sette A., et al. (1994).    Development of high potency universal DR-restricted helper epitopes    by modification of high affinity DR-blocking peptides. Immunity,    1(9): 751-761.-   Ausubel et al. (1987) (1992) (1993) Current Protocols in Molecular    Biology. Wiley Interscience, N.Y.-   Balbi B., Di Stefano A., Capelli A., Balbo P., Lusuardi M., Ioli F.    and Donner C. F. T1 and T2 cytokines in bronchoalveolar lavage of    patients with COPD and with asthma. International Conference of the    American Thoracic Society, 2002.-   Barr I. G. and Mitchell G. F. (1996). ISCOMs (immunostimulating    complexes): the first decade. Immunol. and Cell Biol. 74: 8-25.-   Barnes P. J. (2000). Chronic Obstructive Pulmonary Disease. NEJM    343: 269-80.-   Barnes P. J. (2001). New treatments for chronic obstructive    pulmonary disease. Curr Opin Pharmacology, 1: 217-222.-   Barnes P. J. (2002). New treatments for COPD. Nature reviews Drug    discovery, 1: 437-446.-   Barnes P J. (2003). New concepts in chronic obstructive pulmonary    disease. Annu. Rev. Med., 54:113-29.-   Baughman R. P. (2002). Therapeutic options for sarcoidosis: new and    old. Curr Opin Pulm Med, 8: 464-469.-   Billiau A. (1996) Interferon-γ: biology and role in pathogenesis.    Advances in Immunology 62: 61-130.-   Campbell A. M. (1984) Monoclonal Antibody Technology Techniques in    Biochemistry and Molecular Biology, Elsevier Science Publishers,    Amsterdam, The Netherlands.-   Chackerian B., Lenz P., Lowy D. R. and Schiller J. T. (2002).    Determinants of autoantibody induction by conjugated Papillomavirus    virus-like particles. J Immunol, 169: 6120-6126.-   Ciapponi L., Maione D., Scoumanne A., Costa P., Hansen M. B.,    Svenson M., Bendtzen K., Alonzi T., Paonessa G., Cortese R.,    Ciliberto G. and Savino R. (1997). Induction of interleukin-6 (IL-6)    autoantibodies through vaccination with an engineered IL-6 receptor    antagonist. Nature Biotechnol, 15: 997-1001.-   Coligan J. E., Kruisbeek A. M., D. H. Margulies D. H. (1992) (1993)    Current Protocols in Immunology, Green Publishing Association and    Wiley Interscience, N.Y.-   Contreras C. E., Ploton I. N., Siliciano R. F., Karp C. L.,    Viscidi R. and Kumar N. (1998). Mapping of specific and promiscuous    HLA-DR-restricted T-cell epitopes on the Plasmodium falciparum    27-kilodalton sexual stage-specific antigen Infect Immun, 66(8):    3579-90.-   Casolaro M A, Bernaudin J F, Saltini C, Ferrans V J, Crystal R    G.(1988). Accumulation of Langerhans' cells on the epithelial    surface of the lower respiratory tract in normal subjects in    association with cigarette smoking. Am Rev Respir Dis, 137:406-11.-   Dalum I., Jensen M. R., Hindersson P., Elsner H. I., Mouritsen S.    (1996). Breaking of B cell tolerance toward a highly conserved self    protein. J Immunol 157(11): 4796-804.-   Dalum I., Butler D. M., Jensen M. R., Hindersson P., Steinaa L.,    Waterston A. M., Grell S. N., Feldmann M., Elsner H. I. and    Mouritsen S. (1999). Therapeutic antibodies elicited by    immmunization against TNF-α. Nature Biotechnol, 17: 666-669.-   deBoer W. I. (2002). Cytokines and therapy in COPD. CHEST,    121:209S-218S.-   Donnelly J. J., Ulmer J. B., and Liu M. A. (1997a). DNA vaccines.    Life Sci., 60(3): 163-172.-   Donnelly J. J., Ulmer J. B., Shiver J. W. and Liu M. A. (1997). DNA    vaccines. Annu. Rev. Immunol., 15: 617-648.-   Doolan D. L., Southwood S., Chesnut R., Appela E., Gomez E.,    Richards A., Higashimoto Y. I., Maewal A., Sidney J., Gramzinski R.    A., Mason C., Koech D., Hoffman S. L. and Sette A. (2000).    HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum    pre-erythrocytic-stage antigens restricted by multiple HLA alleles.    J Immunol, 165:1123-1137.-   Dustin M. L., Singer K. H., Tuck D. T. and T. A. Springer (1988)    Adhesion of T lymphoblasts to epidermal keratinocytes is regulated    by interferon-γ and is mediated by intercellular adhesion molecule 1    (ICAM-1). J. Exp. Med. 167: 1323-1340.-   Fernando G. J., Tindle R. W. and Frazer I. H. (1995). T-helper    epitopes of the E7 transforming protein of cervical cancer    associated human papillomavirus type 18 (HPV18). Virus Res,    36(1):1-13.-   Freedman A. S., Freeman G. J., Rhynhart K. and L. M. Nadler (1991)    Selective induction of B7/BB-1 on interferon-gamma stimulated    monocytes: a potential mechanism for amplification of T cell    activation through the CD28 pathway. Cell. Immunol 137:429-437.-   Friedl-Hajek R., Spangfort M. D., Schou C., Breiteneder H., Yssel H.    and Joost van Neerven R. J. (1999). Identification of a highly    promiscuous and an HLA-allele specific T-cell epitope in the birch    major allergen Bet v 1: HLA restriction, epitope mapping and TCR    sequence comparisons. Clin Exp Allergy, 29(4): 478-87.-   Gaudebout P., Zeliszewski D., Golvano J. J., Pignal C., Le Gac S.,    Borras-Cuesta F. and Sterkers G. (1997). Binding analysis of 95 HIV    gp120 peptides to HLA-DR1101 and -DR0401 evidenced many HLA-class II    binding regions on gp120 and suggested several promiscuous regions.    J Acquir Immune Defic Syndr Hum Retrovirol, 14(2): 91-101.-   Gish W. and States D. J. (1993). Identification of protein coding    regions by database similarity search. Nature Genet, 3:266-272.-   Gonzalez S., Alvarez A., Caballero E., Vina L., Guillen G. and    Silva R. (2000). P64k meningococcal protein as immunological carrier    for week immunogens. Scand J Immunol, 52(2): 113-116.-   Harlow E., David L. et. al. (1988) Antibodies: A Laboratory Manual,    Cold Spring Harbor Publications.-   Hautamaki R. D., Kobayashi D. K., Senior R. M. and Shapiro S. D.    (1997). Requirement for macrophage elastase for cigarette    smoke-induced emphysema in mice. Science, 277(5334):2002-4.-   Holliger P., T. Prospero, and G. Winter (1993) Diabodies: small    bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci.    USA 90(14):6444.-   Hurle M. R. and M. Gross (1994) Protein engineering techniques for    antibody humanization. Curr. Opin. Biotech 5:428-433.-   Iliades P., Kortt A. A., and P. Hudson (1997) Triabodies: single    chain Fv fragments without linker form trivalent trimers. FEBS    Letters 409:437-441.-   Kaplan N. M., Palmer B. F. and Weissler J. C. (2000). Syndromes of    Severe Asthma Am Med Sci, 319(3): 166-176.-   Karlsson R., Michaelsson A. and Mattsson L. (1991). Kinetic analysis    of monoclonal antibody-antigen interactions with a new biosensor    based analytical system. J Immunol Methods, 145: 129-240.-   Keatings V M, Collins P D, Scott D M, Barnes P J. 1996. Differences    in interleukin-8 and tumor necrosis factor-α in induced sputum from    patients with chronic obstructive pulmonary disease or asthma.    Am. J. Resp. Crit. Care Med. 153:530-34-   Kilgus J., Jardetzky T., Gorga J. C., Trzeciak A., Gillessen D. and    Sinigaglia F. (1991). Analysis of the permissive association of a    malaria T cell epitope with DR molecules. J Immunol, 146(1): 307-15.-   Kim S. K., Ragupathi G., Capello S., Kagan E., and Livingston P. O.    (2001). Effect of immunological adjuvant combinations on the    antibody and T cell repsonse to vaccination with MUC1-KLH and    GD3-KLH conjugates. Vaccine, 19: 530-537.-   Kim Y -M. and Son K. (1996). A nitric oxide production assay for    interferonγ. J Immunol Methods, 198:203-209.-   Kobayashi H., Wood M., Song Y. Appella E. and Celis E. (2000).    Defining promiscuous MHC class II helper T-cell epitopes for the    HER2/neu tumor antigen. Cancer Res, 60(18): 5228-36.-   Kortt A., Lab M., Oddie G., Gruen C., Burns J., Pearce L., Atwell    J., McCoy A., Howlet G., Metzger D., Webster R. and P. Hudson    (1997). Single-chain Fv fragments of anti-neuramidase antibody NC10    containing five- and ten-residue linkers form dimers and with    zero-residue linker a trimer. Prot. Eng. 10:423-.-   Köhler G. and C. Milstein (1975) Continuous cultures of fused cells    secreting antibody of predefined specificity. Nature 256:495.-   Launois P., Deleys R., Niang M. N., Drowart A., Andrien M., Dierckx    P., Cartel J. L., Sarthou J. L., Van Vooren J. P. and Hyugen K.    (1994). T-cell-epitope mapping of the major secreted mycobacterial    antigen Ag85A in tuberculosis and leprosy. Infect Immun, 62(9):    3679-87.-   Leckie M. J., Bryan S. A., Hansel T. T. and Barnes P. J. (2000).    Novel therapy for COPD. Exp Opin Invest Drugs, 9: 3-23.-   Lethbridge M. W. G, Allenby M. I., Jensen M. W., Kemeny D. M. and    O'Connor B. J. (2002). Airway T lymphocytes expressing IFN-γ are    reduced in smokers with chronic obstructive pulmonary disease    (COPD). Annual Congress 2002 of the European Respiratory    Society-Stockholm.-   Lewis J. A. (1995). A sensitive bioassay for interferons. J Immumol    Methods, 185: 9-17.-   Li Y., Wang M -N., Li H., King K. D., Bassi R., Sun H., Santiago A,    Hooper A. T., Bohlen P. and Hicklin D. J. (2002). Active    immunization against the vascular endothelial growth factor receptor    flk1 inhibits tumor angiogenesis and metastasis. J Exp Med, 195(12):    1575-1584.-   Lorsbach R. B., Murphy W. J., Lowenstein C. .J, Snyder S. H.    and S. W. Russel (1993) Expression of the nitric oxide synthase gene    in mouse macrophages activated for tumor cell killing. J. Biol.    Chem. 268: 1908-1913.-   Lowenadler B. and Lycke N. (1994). Fusion proteins with heterologous    T helper epitopes. Recombinant E. coli heat-stable enterotoxin    proteins. Int Rev Immunol, 11(2): 103-11.-   Majori M., Corradi M., Caminati A., Giancarlo C., Bertacco S. and    Pesci A. (1999). Predominant TH1 cytokine pattern in peripheral    blood from patients with COPD. J Allergy Clin Immunol, 103:458-62.-   Mannino D. M. (2002). COPD: Epidemiology, prevalence, morbidity and    mortality, and disease heterogeneteity. CHEST, 121: 121S-126S.-   Malmqvist M. (1999). BIACORE: an affinity biosensor system for    characterization of biomolecular interactions. Biochemical Society    Transactions, Vol 27.-   Meager A. (2002). Biological assays for interferons. J Immunol    Methods, 261 (1-2): 21-36.-   Mendez M. J., Green L. L., Corvalan J. R., Jia X. C.,    Maynard-Currie C. E., Yang X. D., Gallo M. L., Louie D. M., Lee D.    V., Erickson K. L., Luna J., Roy C. M., Abderrahim H., Kirshenbaum    F., Noguchi M., Smith D. H., Fukushima A., Hales J. F., Klapholz S.,    Finer M. H., Davis C. G., Zsebo K. M. and Jakobovits A. (1997).    Functional transplant of megabase human immunoglobulin loci    recapitulates human antibody response in mice. Nat Genet,    15(2):146-56.-   Möllers M., Aries S. P., Drömann D., Mascher B., Braun J. and    Dalhoff K. (2001). Intracellular cytokine repertoire in different T    cell subsets from patients with sarcoidosis. Thorax, 56: 487-493.-   Morein B. et al. (1995). Clin Immunother. 3:461-475.-   Muller R. (1983) Determination of affinity and specificity of    anti-hapten antibodies by competitive radioimmunoassay. Methods    Enzymol. 92: 589-601.-   Mustafa A. S., Shaban F. A., Abal A. T., Al-attiyah R., Wiker H. G.,    Lundin K. E., Oftung F. and Huygen K. (2000). Identification and HLA    restriction of naturally derived Th1-cell epitopes from the secreted    Mycobacterium tuberculosis antigen 85B recognized by    antigen-specific human CD4+ T-cell lines. Infect. Immun., July:    3933-3940.-   NIH, National heart, lung and blood institute (1995). Sarcoidosis.    NIH publication No. 95-3093, reprinted July 1995.-   NIH, National heart, lung and blood institute (1997). The Lungs in    health and disease. NIH Publication No. 97-3279, August 1997.-   NHLBI/WHO. Executive Summary, Global Strategy for the diagnosis,    management and prevention of chronic obstructive pulmonary disease.    Workshop Report. Pace J. L., Russell S. W., Torres B. A.,    Johnson H. M. and P. W. Gray (1983) Recombinant mouse γ interferon    induces the priming step in macrophage activation for tumor cell    killing. J. Immunol. 130: 2011-2013.-   Oudijk E -JD, Lammers J -W J, Koenderman L. (2003) Systemic    inflammation in chronic obstructive pulmonary disease. Eur Respir J,    22(S46): 5s-13s-   Papiris S., Kotanidou A., Malagar K. and Roussos C. (2002). Clinical    review: severe asthma. Crit Care, 6(1): 3044.-   Pauwels R A, Buist A S, Calverley P M, Jenkins C R, Hurd S S. (2001)    Global strategy for the diagnosis, management, and prevention of    chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative    for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J    Respir Crit Care Med, 163:1256-76.-   Retamales I, Elliott W M, Meshi B, Coxson H O, Pare P D, Sciurba F    C, et al. (2001). Amplification of inflammation in emphysema and its    association with latent adenoviral infection. Amer J Respir Crit    Care Med, 164: 469-473.-   Poljak R. J. (1994) Production and structure of diabodies. Structure    2:1121-1123.-   Richard M., Grencis R. K., Humphreys N. E., Renauld J -C. and Van    Snick J. (2000). Anti-IL-9 vaccination prevents worm expulsion and    blood eosinophilia in Trichuris muris-infected mice. PNAS, 97(2):    767-772.-   Roguska M. A., Pedersen J. T., Keddy C. A., Henry A. H., Searle S.    J., Lambert J. M., Goldmacher V. S., Blättler W. A., Rees A. R.;    and B. C. Guild (1994) Humanization of murine monoclonal antibodies    through variable domain resurfacing. Proc. Natl. Acad. Sci. USA 91:    969-973.-   Rossman M. D. (2001). Chronic Beryllium Disease: A hypersensitivity    disorder. Appl Occup Environm Hygiene, 16(5): 615-618.-   Sad S., Rao K., Arora R., Talwar G. P. and Raghupathy R. (1992).    Bypass of carrier-induced epitope specific suppression using a    T-helper epitope. Immunology, 76: 599-603.-   Sangiuolo F., D'Apice M. R., Bruscia E., Lucidi V. and Novelli G.    (2002). Towards the pharmacogenomics of cystic fibrosis.    Pharmacogenomics, 3(1): 75-87.-   Saltini C. (2001). Beryllium Disease. Am J Med Sci, 321(1): 89-98.-   Shigehara K., Shijubo N. Obmichi M., Tkahashi R., Kon S., Okamura    H., Kurimoto M., Hiraga Y., Tatsumo T., Abe S. and Sato N. (2001).    IL-12 and IL-18 are increased and stimulate IFN-γ production in    sarcoid lungs. J Immunol 166: 642-649.-   Snapper C. M. and W. E. Paul (1987) Interferon-γ and B cell    stimulatory factor-1 reciprocally regulate Ig isotype production.    Science 236: 944-947.-   Steinman R. M., Noguiera N., Witmer M. D., Tydings J. G. and I. S.    Mellman (1980) Lymphokine enhances the expression and synthesis of    Ia antigens on cultural mouse peritoneal macrophages. J. exp. Med.    152: 1248-1261.-   Stirling R. G. and Chung K. F. (2001). Severe Asthma: Definition and    mechanisms. Allergy, 56: 825-840.-   Storni T., Lechner F., Erdmann I., Bächi T., Jegerlehner A., Dumrese    T., Kündig T. M., Ruedl C. and Bachmann M. F. (2002). Critical role    for activation of antigen-presenting cells in priming of cytotoxic T    cell responses after vaccination with virus-like particles. J    Immunol, 168: 2880-2886.-   Talwar G. P. (1999). Vaccines and passive immunological approaches    for the control of fertility and hormone-dependent cancers.    Immunological Reviews, 171: 173-192.-   Udono H. and Srivastava P. K. (1993). Heat shock protein    70-associated peptides elicit specific cancer immunity. J Exp Med,    178(4): 1391-6.-   Verneresson M., Ledin A., Johansson J. and Hellman L. (2002).    Generation of therapeutic antibody responses against IgE through    vaccination. FASEB J. 16: 875-877.-   Wang Z., Zheng T., Zhu Z., Homer R. J., Riese R., Chapman H. A.,    Shapiro S. D. and Elias J. A. (2000). Interferon γ induction of    pulmonary emphysema in the adult murine lung. J Exp Med, 192(11):    1587-1599.-   Wenzel S. E., Szefler S. J., Leung D. Y., Sloan S. I., Rex M. D. and    Martin R. J. (1997). Bronchoscopic evaluation of severe asthma.    Persistent inflammation associated with high dose glucocorticoids.    Am J Respir Crit Care Med, 156(3 Pt 1):737-43.-   Zhu Z., Zapata G., Shalaby R., Snedecor B., Chen H. and P.    Carter (1996) High level secretion of a humanized bispecific diabody    from Escherichia coli. Biotechnology 14:192-196.

1. Use of an IFNγ neutralizing molecule for the manufacture of amedicament for preventing or treating a T1 inflammatory lung disease. 2.Use according to claim 1 wherein said T1 inflammatory lung disease isselected from the group consisting of COPD, emphysema, chronicbronchitis, bronchiolitis, severe asthma, sarcoidosis, berylliosis andcystic fibrosis.
 3. Use according to claims 1-2, wherein said moleculeis an anti-IFNγ antibody or a fragment thereof.
 4. Use according toclaim 3, wherein said antibody is a monoclonal antibody.
 5. Useaccording to claim 4, wherein said antibody is the antibody D9D10 or afragment thereof.
 6. Use according to claim 3, wherein said antibody isa humanized monoclonal antibody.
 7. Use according to claim 6, whereinsaid antibody is a humanized D9D10 antibody.
 8. Use of immunogenic IFNγfor the manufacture of a medicament for preventing or treating a T1inflammatory lung disease.
 9. Use according to claim 8 wherein said T1inflammatory lung disease is selected from the group consisting of COPD,emphysema, chronic bronchitis, bronchiolitis, severe asthma,sarcoidosis, berylliosis and cystic fibrosis.
 10. Use according to claim8, wherein immunogenic IFNγ is a modified IFNγ.
 11. Use according toclaim 10, wherein the IFNγ protein is modified to immunogenic IFNγ bycrosslinking IFNγ to an immunogenic non-self carrier protein.
 12. Useaccording to claim 10, wherein the IFNγ protein is modified toimmunogenic IFNγ by introducing at least one foreign T-cell epitope byinsertion and/or substitution and/or addition and/or conjugation. 13.Use according to claim 8, wherein immunogenic IFNγ is a non-self IFNγadministered with or without adjuvant.
 14. Use according to claim 8,wherein immunogenic IFNγ is autologous IFNγ in combination with anadjuvant.
 15. Use of at least one autologous antigen presenting cellloaded with IFNγ for the manufacture of a medicament for preventing ortreating a T1 inflammatory lung disease.
 16. Use according to claim 16,wherein the antigen presenting cell is a dendritic cell.